An improved suppression subtractive hybridization technique to develop species-specific repetitive sequences from Erianthus arundinaceus (Saccharum complex)

Abstract

BackgroundSugarcane has recently attracted increased attention for its potential as a source of bioethanol and methane. However, a narrow genetic base has limited germplasm enhancement of sugarcane. Erianthus arundinaceus is an important wild genetic resource that has many excellent traits for improving cultivated sugarcane via wide hybridization. Species-specific repetitive sequences are useful for identifying genome components and investigating chromosome inheritance in noblization between sugarcane and E. arundinaceus. Here, suppression subtractive hybridization (SSH) targeting E. arundinaceus-specific repetitive sequences was performed. The five critical components of the SSH reaction system, including enzyme digestion of genomic DNA (gDNA), adapters, digested gDNA concentrations, primer concentrations, and LA Taq polymerase concentrations, were improved using a stepwise optimization method to establish a SSH system suitable for obtaining E. arundinaceus-specific gDNA fragments.ResultsSpecificity of up to 85.42% was confirmed for the SSH method as measured by reverse dot blot (RDB) of an E. arundinaceus subtractive library. Furthermore, various repetitive sequences were obtained from the E. arundinaceus subtractive library via fluorescence in situ hybridization (FISH), including subtelomeric and centromeric regions. EaCEN2-166F/R and EaSUB1-127F/R primers were then designed as species-specific markers to accurately validate E. arundinaceus authenticity.ConclusionsThis is the first report that E. arundinaceus-specific repetitive sequences were obtained via an improved SSH method. These results suggested that this novel SSH system could facilitate screening of species-specific repetitive sequences for species identification and provide a basis for development of similar applications for other plant species.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1471-6) contains supplementary material, which is available to authorized users.

Deep sequencing of suppression subtractive library identifies differentially expressed transcripts of Saccharum spontaneum exposed to salinity stress

“…hybrids ) varieties are the hybrids of S. officinarum and S. spontaneum , in which S. spontaneum is the wild type parent and a highly versatile species with many phenotypes, capable of growing under different biotic and abiotic stress conditions. S. spontaneum is a source of resistance genes such as cold, drought, salinity, high, and low temperature tolerance (Yang et al, 2020; Yu et al, 2018). Salt stress affects sugarcane in two different phases.…”

Chromosomal Characterization of Tripidium arundinaceum Revealed by Oligo-FISH

“…Repetitive DNA probes are the FISH probes used widely in plant genome research, however, such probes always show a varied signal among different species as a result of the instability of genome, so that they cannot be used for cytogenetics research [22]. In T. arundinaceum, Yu et al [23] screened many repetitive sequences; unfortunately, these repeats showed a diversity site that cannot be used as a stable marker for chromosome identification. By comparison, recently, the FISH probe based on single copy sequences designed from genome sequences has provided us a more universal and stable marker for cytological study in plants [15,24].…”

Efficient anchoring of Erianthus arundinaceus chromatin introgressed into sugarcane by specific molecular markers

“…Compared with conventional methods, SSH offers a simple, rapid, and affordable high-throughput screening method for separating DNA sequences that distinguish two closely related species. Although SSH only has been applied successfully to separate species-speci c sequences in a few plants, this method had already proved to be capable of screening out a reservoir of species-speci c molecular markers (Ge et al 2012;Li et al 2004;Yu et al 2018). Notably, accurate identi cation of putative progeny hinges on the adequate molecular markers covering the whole genome.…”

“…Notably, accurate identi cation of putative progeny hinges on the adequate molecular markers covering the whole genome. In SSH array, the e cient ampli cation of species-speci c sequences by PCR is thank to that those short and high copy number fragments tend to be ampli ed in preference to larger and lower copy number ones (Yu et al 2018). Highcopy-number repetitive sequences comprise most eukaryotic genomes where they are major contributors to genome evolution (Heslop-Harrison and Schwarzacher 2011; Naito et al 2009).…”